Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02–2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

Introduction dark cycle at animal center, Faculty of Medicine, Chulalongkorn University. Mice were kept in the same cage for the determination of several parameters in chili-administered model, while some mice were kept in different cages in each experimental group for the microbiota analysis.
To compare the result between 6 independent groups with one-way analysis of variance (ANOVA), we use G-power program to calculate sample size per group. Therefore, a minimum number of mice (8 mice per group) were used to obtain the proper statistical test and the significance was determined by p-value < 0.05. Then, a total of 48 mice were randomized into several groups for the determination of several parameters in chili-administered model and the feces from some mice were used for the fecal microbiome analysis. For investigating the effect of Lactobacillus rhamnosus in chili-administered model, mice were randomly divided into 6 groups: the control phosphate buffer solution (PBS) group (n = 8) without chili administration, the control L. rhamnosus 34 (L34) group (n = 8), the control L. rhamnosus GG (LGG) group (n = 8) and the chili-extract administered group treated with PBS (n = 8), L34 (n = 8), and LGG (n = 8) which received at 1 × 10 8 colonies forming unit (CFU) of Lactobacilli. For chili extracts, 40 g of Thai chili was diluted in 250 mL of absolute ethanol before autoclave at 121˚C for 15 min following a previous publication [34]. After that, the samples were subjected to filtration twice with Whatman filter paper (number 42) (GE Healthcare, Chicago, IL, USA) and dried in hot air oven at 55˚C (Shel Lab, Cornelius, OR, USA) for 4 days before suspension by 60 mL sterile water. According to this protocol, the chili extract approximately contains 1 mg of capsaicin per 1 mL of the solution [34]. Then, the chili extract at 0.5 mL (approximately 50 mg capsaicin/ dose) or phosphate buffer solution (PBS) control was orally administered in mice once a day at 8:00 AM. In parallel, the probiotics, including Lactobacillus rhamnosus L34 (L34) (Chulalongkorn University, Bangkok, Thailand) or Lactobacillus rhamnosus GG (LGG) (Mead-Johnson, Evansville, IN, USA) at 1 × 10 8 colonies forming unit (CFU) in 0.3 mL PBS or PBS alone were orally administered once a day at 16:00 PM. Mice were observed and monitored daily for body weight, stool consistency and feces from each group of mice were collected before sacrifice for microbiome analysis. After 14 days administration, mice were euthanized with cardiac puncture under isoflurane anesthesia and mouse samples (blood, and colon tissue) were collected. Serum and colon tissue were snap frozen in liquid nitrogen and kept in -80˚C before use. The stool consistency was semi-quantitatively evaluated as "the stool consistency index" using the following score; 0, normal; 1, soft; 2, loose and 3, diarrhea, as previously published [35].

Fecal microbiome analysis
Feces from each mouse (0.25 g per mouse; 3 mice per group) from different cages in each experimental group were collected for the microbiota analysis following a previous report [40]. Of note, mice in the same groups were housed in different cages because co-housing might induce similar gut microbiota within the same cage. In short, metagenomic DNA was extracted from individual mice by DNeasy PowerSoil Kit (Qiagen, Maryland, USA). The quality and concentration of the extracted DNA were measured by agarose gel electrophoresis and nanodrop spectrophotometry. Libraries of the V4 hypervariable region of 16S rRNA gene were amplified by polymerase chain reaction (PCR) using Universal prokaryotic primers 515F (forward; 5 0 -GTGCCAGCMGCCGCGGTAA-3 0 ) and 806R (reverse; 5 0 -GGACTACHVGGGTWT CTAAT-3 0 ), modified with the Illumina adapter and Golay barcode sequences in Miseq300 platform (Illumina, San Diego, CA, USA). The raw sequences and operational taxonomic unit (OTU) were classified following Mothur's standard operating platform [41,42]. The non-metric multidimensional scaling (NMDS), the distance-based ordination method, was performed based on the Bray-Curtis dissimilarity. The 16S rDNA sequences in this study were deposited in an NCBI open access Sequence Read Archive database with accession number PRJNA776693.

Human fecal samples
Feces of the healthy volunteers were collected at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand following the approved protocol by the Ethical Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB No. 130/62), according to the Declaration of Helsinki, with the written informed consent from each individual volunteer. The inclusion criteria of the volunteers were i) adults between 18-65 years old without neither underlying diseases nor any current medications and ii) ingestion of Thai chili containing diet at least 1 meal per day. The exclusion criteria were i) underlying diseases (hypertension, diabetes, liver diseases, and kidney injury), ii) any medications, including antibiotics and health supplements within 1 month of the recruitment, and iii) ingestion of any products containing probiotics (for example; Yogurt, Kimchi and pickled fish) within 2 weeks. Then, the volunteers were orally once daily administered with L. rhamnosus L34 (Greater Pharma co., Bang Phlat, Bangkok, Thailand) or L. rhamnosus GG (Mead-Johnson) at 1 × 10 9 CFU/dose. To determine the abundance of fecal Lactobacilli of the healthy volunteer, real-time polymerase chain reaction (PCR) was performed on fecal contents at the baseline (3 days before probiotic administration; 0 time-point (D0)) and after several days of administration, including 3 (D3), 7 (D7) and 20 (D20) days, and after stop the probiotics for 3 days (D23) and 7 days (D30) to explore rate of the probiotic reduction. The total DNAs were extracted by a QIAamp fast DNA Stool Mini Kit (Qiagen, Hiden, Germany) following manufacturer's instructions with the primers for variable regions of 16S rRNA gene sequence of L. rhamnosus; rham (forward; 5 0 -TGCAT CTTGATTTAATTTTG-3 0 ) and Y2 (reverse; 5 0 -CCCACTGCTGCCTCCCGTAGGAGT-3 0 ) [43]. The amplicon was approximately 290 base pairs (bp) and the genome size of L. rhamnosus (also designated as LR ATCC 53103) was 3,005,051 bp [44]. Bacterial genome is approximately 1.98 × 10 9 g/mol and contains 6.02 × 10 23 molecules/mol. One bacterium corresponds to 3.3 fg of DNA. The constructive of standard curve was generated by the QuantStudio™ Design & Analysis Software v1.4.3 (Thermo Fisher Scientific) using 10-fold serial dilution (6.6 fg to 660 pg) with bacterial concentrations ranging from of 2 to 2 × 10 5 bacteria. In parallel, the quantification of fecal bacteria was indicated by real-time PCR which represented by cycle threshold (Ct value). Real-time PCR was performed in a QuantStudio™ Design & Analysis Software v1.4.3 with primers are as followed; total Gram negative bacteria (16S rRNA Gram neg.) (forward; 5 0 -GGAGGAAGGTGGGGATGACG-3 0 , reverse; 5 0 -ATGGTGTGACGGGCGGTGTG-3 0 ), [48], Akkermansia (AM) (forward; 5 0 -CAGCACGTGAAGGTGGGGAC-3 0 , reverse; 5 0 -CCTTGCGGTTGGCTTCAGAT-3 0 ) [49], and total fungi (ITS) (forward; 5 0 -TCCGTAGGTGAACCTGCGG-3 0 and reverse; 5 0 -TCCTCC GCTTATTGATATGC-3 0 ) [50].

Cell viability test of capsaicin-activated enterocytes
Because of the known cytotoxicity of capsaicin [12,13] (a main substance responsible for the hot and spicy taste in the chili), capsaicin (305.41 g/mol molecular weight; Sigma-Aldrich, St. Louis, MO, USA) were tested with enterocytes (Caco-2 and HT-29 cell lines). As such, the human colorectal adenocarcinoma cells, Caco-2 (ATCC HTB-37) and HT-29 (ATCC HTB-38), from the American Type Culture Collection (Manassas, VA, USA) were maintained in supplemented Dulbecco's modified Eagle medium (DMEM) and McCoy's 5a modified medium, respectively, at 37˚C under 5% CO 2 and sub-cultured before use in the experiments. Then, capsaicin in the different concentrations (0.02, 0.2 and 2 mM) was incubated with the enterocytes for 24 h before the determination of cell viability using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) solution (Thermo Fisher Scientific, Rockford, IL, USA) [38]. The activated cells were incubated with 0.5 mg/mL of MTT solution for 2 h at 37˚C in the dark and diluted by dimethyl sulfoxide (DMSO; Thermo Fisher Scientific) before measurement with a Varioskan Flash microplate reader at absorbance of optical density (OD) 570 nm.

Proinflammatory activation in enterocytes
Because capsaicin induces diarrhea [9] possibly through capsaicin-induced gut hypermobility [10,11] and cytotoxicity [12,13] (especially with the presence of microbial molecules), capsaicin alone or with lipopolysaccharide (LPS), a major component of Gram-negative bacteria in gut, with or without (1!3)-β-D-glucan (BG), a major component of fungi in gut, were incubated with the enterocytes with and without the Lactobacillus condition media (LCM). For LCM preparation, L. rhamnosus L34 or LGG at an OD600 of 0.1 were incubated anaerobically for 48 h before supernatant collection by centrifugation and filtration (0.22-μm membrane filter) (Minisart; Sartorius Stedim Biotech GmbH, Göttingen, Germany). After that, cell-free supernatant of the samples (500 μL) was concentrated by speed vacuum drying at 40˚C for 3 h (Savant Instruments, Farmingdale, NY), resuspended in an equal volume of DMEM or McCoy's 5a modified medium for testing in Caco-2 or HT-29 cells, respectively, and stored at -20˚C until use. Then, capsaicin (Sigma-Aldrich) at 0.02 mM alone or with LPS from E. coli O26:B6 (Sigma-Aldrich) at 100 ng/mL with or without BG, using whole glucan particle (WGP) that was purified from Saccharomyces cerevisiae (Biothera, Eagan, MN), at 100 μg/mL were incubated with the enterocytes with or without 5% (vol/vol) LCM (each strain) (the total volume was adjusted into 200 μL/well by the culture media) for 24 h before determination the level of IL-8 by using a Human CXCL8/IL-8 ELISA kit (Quantikine immunoassay; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Transepithelial electrical resistance (TEER) and enterocyte permeability
The integrity of monolayer enterocytes in different conditions was determined by TEER using Caco-2 cells, but not HT-29 cells due to a limited ability of monolayer growth of HT-29 cells [51]. Caco-2 cells (ATCC HTB-37) at 5 × 10 4 cells per well were seeded onto the upper compartment of 24-well Boyden chamber trans-well plate using DMEM-high glucose supplemented with 20% fetal bovine serum (FBS), 1% HEPES, 1% sodium pyruvate, and 1.3% Penicillin/Streptomycin for 15 days with daily media replacement to establish the confluent monolayer. After that, capsaicin (Sigma-Aldrich) at 0.02 and 0.2 mM with or without 5% (vol/ vol) LCM of L. rhamnosus L34 or LGG were incubated at 37˚C under 5% CO 2 for 24 h. Subsequently, TEER was measured by an EMOM 2 Epithelial Voltohmmeter (World Precision Instruments Inc., Sarasota, FL, USA) by placing the electrodes in supernatant at basolateral and apical chamber. The TEER value in media culture without cells was used as a blank and was subtracted from all measurements. The unit of TEER was ohm (O) × cm 2 . In parallel, 5 μL of FITC-dextran (4.4 kDa) (Sigma-Aldrich) at 10 mg/mL was added to the apical side of the trans-well chamber with 24 h stimulated Caco-2 cells. Then, FITC-dextran from the basolateral side of the trans-well plate was measured at 1 h after incubation using Fluorospectrometer (NanoDrop 3300) (ThermoFisher Scientific) as modified from the published protocols [52][53][54]. The concentration of FITC-dextran from the basolateral side represents the severity of permeability defect of Caco-2 cells.

Capsaicin bactericidal effect against the probiotics
Due to the possible bactericidal activity of capsaicin against the probiotics [15], capsaicin (Sigma-Aldrich) at 20 mM in different concentrations (0.2, 2, and 20 mM) or medium control (De Man, Rogosa and Sharpe; MRS) were incubated with L. rhamnosus L34 (Chulalongkorn University) or L. rhamnosus GG (Mead-Johnson) at 3.0 × 10 7 CFU/mL for 24 h before determination of bacterial abundance as previously described [55]. Briefly, L. rhamnosus at an OD600 of 0.1 (3.0 × 10 7 CFU/mL) in MRS broth with or without capsaicin (Sigma-Aldrich) at 20 mM were incubated anaerobically for 24 h. After incubation, the optical density of each culture was determined at 600 nm (OD600) by spectrophotometer (Bio-Rad Smart Spec Plus, Bio-Rad Laboratories Inc, Hercules, CA, USA) to calculate bacterial number, and then 10-fold serially diluted in MRS broth, and cultured as stated previously for 48 h for bacterial enumeration.
Extracellular flux analysis. Because of the known influence on cell energy of capsaicin in cancer cells [56], the energy metabolism profiles of enterocytes (HT-29 cells) [57] with the estimation of glycolysis and mitochondrial oxidative phosphorylation through extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, were performed by the Seahorse XF Analyzers (Agilent, Santa Clara, CA, USA) as previously described [28,29,[58][59][60][61][62]. In brief, HT-29 cells (1 × 10 4 cells/ well) were grown and stimulated with capsaicin in with McCoy's 5a modified medium control, with or without LCM of L. rhamnosus L34 or LGG for 24 h in a Seahorse cell culture plate before replacing by Seahorse substrates (glucose, pyruvate, and L-glutamine) (Agilent, 103575-100) in pH 7.4 at 37˚C for 1 h prior to the challenge with different metabolic interference compounds including oligomycin 1.5 μM, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) 1 μM, and rotenone/ antimycin A 0.5 μM according to the manufacturer's instructions. The glycolysis data were analyzed by Seahorse Wave 2.6 software.

Statistical analysis
Mean ± standard error of mean (SEM) was used for data presentation. The difference between groups was examined for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey's analysis or Student's t-test for comparisons of multiple groups or 2 groups, respectively. All statistical analyses were performed with SPSS 11.5 software (SPSS, IL, USA) and GraphPad Prism version 9.0 software (La Jolla, CA, USA). A p-value of < 0.05 was considered statistically significant.

Chili extracts induced loose stool and mild intestinal inflammation, partly through gut dysbiosis
Chili extracts administration for 2 weeks in mice caused loose stool without an alteration in body weight and systemic inflammation (serum TNF-α) (Fig 1A-1C). However, the chili extracts induced gut permeability defect as indicated by FITC-dextran assay and the loss of intestinal tight junction molecules; occludin and ZO-1 (using fluorescent color staining and gene expression analysis), without the enhanced gut translocation of organismal molecules; (1!3)-β-D-glucan (BG) and lipopolysaccharide (LPS) (undetectable), nor colon histological injury (Figs 1D-1J and 2), indicating the limited severity of intestinal injury. Notably, LPS and BG are the major component of Gram-negative bacteria and fungi, respectively, which are the most and the second most abundance organisms, respectively, in gut [63]. Additionally, chili extracts also induced inflammatory cytokines (TNF-α and IL-6) in the colon tissue (Fig 1K  and 1L), which supported a possible enterocyte toxicity from high dose of chili [2,64]. The administration of L. rhamnosus, either L34 or LGG strains, attenuated the local inflammation (cytokines in colon tissue) (Fig 1K and 1L) and improved intestinal tight junction (Fig 1F, 1G, 1I and 1J) but not reduced the severity of gut barrier defect ( Fig 1D) and not altered stool consistency (Fig 1A).
Because of bactericidal activity of capsaicin (a major chili active-component) [65], chiliinduced gut inflammation might, at least in part, be due to gut dysbiosis. Indeed, chili extracts

PLOS ONE
induced gut dysbiosis as indicated by increased Bacteroides, the Gram-negative anaerobes with a possible pathogenesis [51], decreased Firmicutes, the predominant organisms in healthy condition [51], and enhanced fecal total Gram-negative bacteria, a source of LPS (a potent pro-inflammatory inducer) in gut [60][61][62]66], without an alteration in Proteobacteria, the pathogenic Gram-negative aerobes [51] (Fig 3A-3D). However, chili extracts did not alter neither the variety of microorganisms (Chao and Shannon indexes of alpha diversity) nor microbe abundance in gut as the values of the total OTUs (Fig 3E). Administration of L. rhamnosus L34 (L34) or L. rhamnosus GG (LGG) in the chili-extract administered mice reduced Bacteroides, decreased fecal Gram-negative bacteria and increased Firmicutes (Fig 3A-3D). Furthermore, the difference in fecal microbiome was demonstrated by a separation in nonmetric multidimensional scaling (NMDS) of bacteria based on the species taxonomic level (S1A Fig). The major organisms (NMDS analysis) in control PBS group and chili-administered mice were Streptococcus spp. and Butyricicoccus spp., respectively, while various bacteria were indicated in the probiotic-administered groups implying an impact of probiotics on gut bacteria (S1A-S1C Fig).

Capsaicin cytotoxicity on enterocytes and on probiotics, a possible impact of spicy food
Due to the well-known cytotoxicity and mild bactericidal activity of capsaicin [15,55,67,68], capsaicin might directly induce enterocyte injury and reduce probiotics abundance. Indeed, capsaicin concentrations that higher than 0.2 mM reduced cell viability in both Caco-2 and HT-29 enterocytic cells which could be attenuated by Lactobacillus condition media (LCM) from both strains of the probiotics (L34 and LGG) (Fig 4A, 4B). Although capsaicin at 0.02 mM did not reduce cell viability (Fig 4A and 4B), this dose of capsaicin enhanced pro-inflammatory cytokine (IL-8) production that was also attenuated by LCM from both probiotics ( Fig  4C and 4D). Notably, supernatant cytokines were non-detectable in enterocytes (Caco-2 and HT-29) in the control group (cell culture media alone). Additionally, capsaicin at 0.2 mM also worsened the integrity of enterocyte tight junction as indicated by the trans-epithelial electrical resistance (TEER) and trans-epithelial FITC-dextran (on Caco-2 cells) but LCM strengthened the integrity (Fig 4E and 4F). Although capsaicin at 0.2 and 2 mM reduced enterocyte permeability (Fig 4E and 4F) and cause enterocytic cell death (Fig 4A and 4B), respectively, capsaicin in both concentrations did not reduce the abundance of both L34 and LGG (Fig 4G). Capsaicin at a high dose (20 mM) decreased abundance of LGG, but not L34 (Fig 4G), implied a better capsaicin tolerance of L34. In parallel, capsaicin (at 0.2 mM), but not at 0.02 mM, decreased enterocyte cell energy as indicated by the reduced glycolysis activity, but not mitochondrial energy production (Fig 5A-5D), which supported a possible interference of cell energy metabolism by capsaicin as previously mentioned [56]. However, LCM from both L34 and LGG restored glycolysis activity (Fig 5A-5D) that might be associated with the reduced enterocyte inflammatory responses against capsaicin (Fig 4A-4F).
Because L. rhamnosus L34 is derived from Thai population and most of Thai cuisines compose of chili (even with some fruits), L34 might be a strain with a higher tolerance to chili than LGG (the probiotics derived from the Caucasian) [69]. Then, L34, LGG or placebo was administered in healthy Thai volunteers ( Table 1) that consume Thai chili at least a meal per day. However, abundance of Lactobacilli in feces of the volunteers was not different between L34 and LGG after the administration for 3, 7, and 20 days (Fig 6A-6C). The Lactobacilli abundance in the volunteers with placebo was very low or non-detectable in the placebo group and the abundance in the probiotic-administered groups (more than 7 days) were higher than the placebo group but rapidly disappeared within 3 days after stop probiotics (Fig 6A-6D). The  (Fig 6A-6D), despite a better tolerance on the high dose capsaicin of L34 over LGG in vitro (Fig 4G). In some time-points of administration, fecal bacterial abundance (detected by real-time PCR) of the L34-administered volunteers, including Klebsiella, Bacteriodes, Bacteriodes fragilis, and total Gram-negative bacteria, were lower than the LGG group, while fecal abundance of Lactobacilli and total fungi in the L34-administered group were higher than the LGG group ( Fig  6E). Further studies on the impact of heavy spicy foods or other diets on probiotics are interesting.

Discussion
The high dose of chili administration for 2 weeks induced mild enterocyte inflammation and gut permeability defect through chili-induced gut dysbiosis and capsaicin-induced enterocyte cytotoxicity, which could be attenuated by Lactobacillus rhamnosus. Despite several health benefits of capsaicin (a major beneficial component in chili) [70], the substance in a high dose (50 mg/day) might induce an adverse effect as indicated by gut barrier defect from the high dose of chili extracts. Here, 2 weeks administration of Thai chili extracts induced loose stool in all mice supporting the well-known capsaicin induced gut hyper-motility [10,11]. Due to the inadequate time in the intestine for water absorption, gut hyper-motility causes loose stool and diarrhea. Additionally, the chili extracts also induced gut permeability defect that severe enough for the translocation of FITC-dextran, a non-gut absorbable with molecular weight (MW) 4.4 kDa, but was not severe enough for gut translocation of the larger pathogen molecules, including lipopolysaccharide (LPS; MW 10-100 kDa or higher) and (1!3)-β-D-glucan (BG; MW 6-600 kDa or higher) [63]. Notably, passive transport through the healthy intestinal tight junction normally allows gut translocation of only the molecules smaller than 0.6 kDa [63]. Despite the non-translocation of LPS and BG, there was a mild reduction of occludin and ZO-1, the tight junction molecules, after chili administration indicating a possible higher sensitivity of a fluorescent-based detection of gut barrier defect.
Although the anti-inflammatory and anti-oxidant effects of capsaicin are mentioned [6,71,72], the high dose of capsaicin induces neurogenic inflammation and cytotoxicity [73,74]. Here, the dose-related enterocyte toxicity of capsaicin was indicated as the low dose capsaicin (0.02 mM) induced only enterocyte inflammation (IL-8 production) without an effect on cell viability, transepithelial electrical resistance (TEER), and cell energy. However, the pro-inflammatory effect of 0.02 mM capsaicin against enterocytes were enhanced by the presence of LPS and BG. With 0.2 mM capsaicin, there was a reduction in enterocytic cell viability, TEER, and cell glycolysis activity supporting several previous reports [1,14,56,[75][76][77][78][79]. Furthermore, capsaicin in the very high dose (2 mM) reduced enterocyte viability and demonstrated bactericidal effect on L. rhamnosus GG supporting cytotoxicity and bactericidal activity of capsaicin [14,[80][81][82]. Hence, gut barrier defect from the high dose chili extracts might be partly responsible from the direct enterocyte cytotoxicity of capsaicin that induced local inflammation (colon cytokines; TNF-α and IL-6) and loose stool.
High dose chili extracts induced intestinal barrier defect partly through fecal dysbiosis. The fecal dysbiosis is another common cause of intestinal tight junction defect and diarrhea [26] that might be partly associated with the loose stool after chili administration. Accordingly, the chili extracts induced gut dysbiosis as indicated by reduced Firmicutes, the highest abundant level with the average abundance (A-C), relative abundance of bacterial diversity at phylum with graph presentation on Bacteroides, Proteobacteria, Firmicutes, and the fecal Gram-negative bacteria (D) and the alpha diversity by Chao1 index richness estimation and Shannon's index evenness estimation with total operational taxonomic unit (OTUs) (E) are demonstrated. � , p < 0.05; # , p < 0.05.
https://doi.org/10.1371/journal.pone.0261189.g003  [83], and increased Bacteroides, bacteria with possible pathogenicity in some conditions [84], and enhanced total Gram-negative bacteria, the source of LPS in mouse feces. Although capsaicin in a lower dose (8 mg/kg/dose) increases Firmicutes and reduces Bacteroides in mice [85], high dose capsaicin (more than 0.33 mM) demonstrates bactericidal effect against some bacteria, including Firmicutes and Bacteroides [86], that might be responsible for chili-induced fecal dysbiosis are previously reported. As such, the non-metric multidimensional scaling (NMDS) analysis identified 2 different predominant bacterial clusters as following; i) Streptococcus spp. (Family; Streptococcaceae) in the control PBS group and ii) Butyricicoccus spp. (Family; Clostridiaceae) in the chili-administered group. Although both bacteria are possible beneficial microbes in Firmicutes group [87][88][89], the NMDS analysis demonstrated some differences between chili-administration and the control PBS group. Not only bactericidal effect of high dose capsaicin, the intestinal inflammation is also a factor that could directly induce fecal dysbiosis [90] and some of the beneficial microbes (such as Butyricicoccus spp.) might be enhanced to counteract with the dysbiosis. More exploration on Butyricicoccus spp. might be interesting. Meanwhile, NMDS plot among L. rhamnosus (either L34 or LGG) administered groups identified several bacteria in the different directions from the control PBS and the chili-administered groups, implying a possible impact of the probiotics.
On the other hand, there were similar benefits of L. rhamnosus GG and L. rhamnosus L34, despite the capsaicin bactericidal effect on L. rhamnosus GG. As such, the attenuation of gut barrier defect and fecal dysbiosis by several probiotics, including L. rhamnosus, is well-known [28,29,51]. Here, both L. rhamnosus L34 (L34; the Thai-derived probiotics) and L. rhamnosus GG (LGG; the commercially available Caucasian-derived probiotics) attenuated intestinal inflammation, tight junction defect, and fecal dysbiosis. While the NMDS fecal analysis clearly separated between the chili-administration and the control PBS groups, the analysis on feces from mice with probiotics could not be clearly separated to other groups. Both probiotics (L34 and LGG) increased Firmicutes, reduced Bacteroides, and decreased total Gram-negative bacteria when compared with the chili-extract administered mice without probiotics. Although several mechanisms are responsible for probiotics beneficial effects, the anti-inflammatory substances might be one of these mechanisms. Indeed, Lactobacillus condition media (LCM) from both L34 and LGG attenuated several effects of high dose capsaicin stimulation, including cell viability, pro-inflammation, and cell resistance (TEER) which might be associated with a preservation on the glycolysis activity. Indeed, cell energy status of the enterocytes is necessary for the maintenance of several cell activities [91,92]. In parallel, 24 h incubation of 2 mM capsaicin reduced LGG abundance but no effect on L34, suggesting a higher tolerance to the high dose capsaicin of L34. Although the influence of diets on fecal microbiome patterns [22] and antibiotic resistance of probiotic bacteria [93] are well-known, the data on probiotic resistance against some specific diets is still very less.
To further test an impact of capsaicin tolerance of L rhamnosus, probiotics (L34 or LGG) or placebo was administered in healthy volunteers who had spicy foods at least a meal per day. Among these volunteers, the abundance of fecal Lactobacillus in placebo group was very less and the probiotics administration at least 7 days was necessary to sustain the fecal abundance which rapidly decreased within 3 days after stop the probiotics. The alterations of fecal abundance of L34 and LGG after administration were not different, despite the higher tolerance against capsaicin of L34 in vitro. Perhaps, the dose of capsaicin from the regular chili- containing Thai foods was not high enough to show the difference in abundance of L34 and LGG in feces. More studies of the influence of diets on probiotics are interesting.

Conclusion
Thai chili extracts and high dose capsaicin induced gut barrier defect through enterocyte cytotoxicity and bactericidal activity-induced gut dysbiosis which were attenuated by L. rhamnosus probiotics. Although L. rhamnosus L34 (the Thai-derived probiotics) was more tolerance against 2 mM capsaicin than L. rhamnosus GG (Caucasian-derive probiotics), the Lactobacillus fecal abundance in the healthy Thai volunteers with chili ingestion of both strains of probiotics bacteria was non-different. Further tests on the volunteers with heavy chili ingestion are interesting.